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dictyNews Volume 20 Number 02
Dicty News
Electronic Edition
Volume 20, number 2
February 15, 2003
Please submit abstracts of your papers as soon as they have been
accepted for publication by sending them to dicty@northwestern.edu.
Back issues of Dicty-News, the Dicty Reference database and other
useful information is available at DictyBase--http://dictybase.org.
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Abstracts
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DIF-1, an anti-tumor substance found in Dictyostelium discoideum,
inhibits progesterone-induced oocyte maturation in Xenopus laevis
Yuzuru Kuboharaa*, Yoichi Hanaoka, Emi Akaishi, Hisae Kobayashi,
Mineko Maeda, Kohei Hosaka
Biosignal Research Center, Institute for Molecular and Cellular
Regulation (IMCR), Gunma University, Maebashi 371-8512, Japan
European Journal of Pharmacology, In press
Abstract
Differentiation-inducing factor-1 (DIF-1; 1-(3,5-dichloro-2,6-dihydroxy-
4-methoxyphenyl)hexan-1-one) is a putative morphogen that induces stalk-
cell formation in the cellular slime mold Dictyostelium discoideum. DIF-1
has previously been shown to suppress cell growth in mammalian cells. In
this study, we examined the effects of DIF-1 on the progesterone-induced
germinal vesicle breakdown in Xenopus laevis, which is thought to be
mediated by a decrease in intracellular cAMP and the subsequent activation
of mitogen-activated protein kinase (MAPK) and maturation-promoting factor,
a complex of cdc2 and cyclin B, which regulates germinal vesicle breakdown.
DIF-1 at 10-40 micro M inhibited progesterone-induced germinal vesicle
breakdown in de-folliculated oocytes in a dose-dependent manner.
Progesterone-induced cdc2 activation, MAPK activation, and c-Mos
accumulation were inhibited by DIF-1. Furthermore, DIF-1 was found to
inhibit the progesterone-induced cAMP decrease in the oocytes. These
results indicate that DIF-1 inhibits progesterone-induced germinal vesicle
breakdown possibly by blocking the progesterone-induced decrease in
[cAMP]i and the subsequent events in Xenopus oocytes.
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The AP-1 clathrin-adaptor is required for lysosomal enzymes sorting and
biogenesis of the contractile vacuole complex in Dictyostelium cells
Yaya Lefkir,* Benot de Chassey,* Annick Dubois,* Aleksandra Bogdanovic,
Rebecca J. Brady, Olivier Destaing, Franz bruckert, Theresa J.
O'halloran, Pierre Cosson, and Franois Letourneur*#
* Institut de Biologie et Chimie des Protines, UMR5086 - CNRS/Universit
Lyon I 7, Passage du Vercors, 69367 Lyon cedex 07, France; Laboratoire de
Biochimie et Biophysique des Systmes Intgrs, 38054 Grenoble Cedex 9,
France; 241 Patterson Laboratories, Section of Molecular Cell & Developmental
Biology, The University of Texas at Austin, 24th St. and Speedway Avenue,
Austin, TX 78712, USA; Laboratoire de Biologie Molculaire et Cellulaire
/ UMR 5665 Ecole Normale Suprieure de Lyon, 69364 LYON Cedex 07, France;
Universit de Genve, Centre Mdical Universitaire, Dpartement de
Morphologie, CH-1211 Genve 4, Switzerland
Molecular Biology of the Cell, In Press
ABSTRACT
Adaptor protein complexes (AP) are major components of the cytoplasmic
coat found on clathrin-coated vesicles. Here, we report the molecular and
functional characterization of Dictyostelium clathrin-associated AP-1
complex, which in mammalian cells, participates mainly in budding of
clathrin-coated vesicles from the trans-Golgi network (TGN). The g-adaptin
AP-1 subunit was cloned and shown to belong to a Golgi-localized 300kDa
protein complex. Time-lapse analysis of cells expressing g-adaptin tagged
with the green-fluorescent-protein demonstrates the dynamics of AP-1
coated structures leaving the Golgi apparatus and rarely moving towards
the TGN. Targeted disruption of the AP-1 medium chain results in viable
cells displaying a severe growth defect and a delayed developmental cycle
as compared to parental cells. Lysosomal enzymes are constitutively
secreted as precursors, suggesting that protein transport between the
TGN and lysosomes is defective. Whereas endocytic protein markers are
correctly localized to endosomal compartments, morphological and
ultrastructural studies reveal the absence of large endosomal vacuoles
and an increased number of small vacuoles. In addition, the function of
the contractile vacuole complex (CV), an osmoregulatory organelle is
impaired and some CV components are not correctly targeted.
submitted by: Francois Letourneur [f.letourneur@ibcp.fr]
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Title: Analysis of 5 Nucleotidase and Alkaline Phosphatase by Gene
Disruption in Dictyostelium
Charles L. Rutherford*, Danielle F. Overall, Muatasem Ubeidat and
Bradley R. Joyce
Biology Department, Molecular and Cellular Biology Section, Virginia
Polytechnic Institute and State University, Blacksburg, VA 24061-0406, USA
Genesis, In Press
Abstract
In Dictyostelium discoideum a phosphatase with a high pH optimum is known
to increase in activity during cell differentiation and become localized
to a narrow band of cells at the interface of prespore and prestalk cells.
However, it was not clear if this activity is due to a classical alkaline
phosphatase with broad range substrate specificity or to a
5 nucleotidase with high substrate preference for 5 AMP. We attempted
to disrupt the genes encoding these two phosphatase activities in order
to determine if the activity that is localized to the interface region
resides in either of these two proteins. During aggregation of 5nt null
mutants multiple tips formed rather than the normal single tip for each
aggregate. In situ phosphatase activity assays showed that the wt and
the 5nt gene disruption clones had normal phosphatase activity in the
area between prestalk and prespore cell types, while the alp null mutants
did not have activity in this cellular region. Thus, the phosphatase
activity that becomes localized to the interface of the prestalk and
prespore cells is Alkaline Phosphatase.
Submitted by rutherfo@vt.edu
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ADENYLYL CYCLASE LOCALIZATION REGULATES STREAMING DURING
CHEMOTAXIS
Paul W. Kriebel, Valarie A. Barr, and Carole A. Parent*
Laboratory of Cellular and Molecular Biology, National Cancer Institute,
National Institutes of Health
Cell, In press.
We studied the role of the adenylyl cyclase ACA in D. discoideum chemotaxis
and streaming. In this process cells orient themselves in a head to tail
fashion as they are migrating to form aggregates. We show that cells lacking
ACA are capable of moving up a chemoattractant gradient, but are unable to
stream. Imaging of ACA-YFP reveals plasma membrane labeling highly enriched
at the uropod of polarized cells. This localization requires the actin
cytoskeleton but is independent of the regulator CRAC and the effector PKA.
A constitutively active mutant of ACA shows dramatically reduced uropod
enrichment and has severe streaming defects. We propose that the asymmetric
distribution of ACA provides a compartment from which cAMP is secreted to
locally act as a chemoattractant, thereby providing a unique mechanism to
amplify chemical gradients. This could represent a general mechanism that
cells use to amplify chemotactic responses.
submitted by Carole Parent [parentc@helix.nih.gov]
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Characterization of a cAMP-stimulated cAMP phosphodiesterase in
Dictyostelium discoideum
Marcel E. Meima, Karin E. Weening and Pauline Schaap
School of Life Sciences, University of Dundee, MSI/WTB complex, Dow Street,
Dundee DD1 5EH, UK.
SUMMARY
A cyclic nucleotide phosphodiesterase, PdeE, that harbours two cyclic
nucleotide binding motifs and a binuclear Zn2+ binding domain, was
characterized in Dictyostelium. In other eukaryotes, the Dictyostelium
domain shows greatest homology to the 73 kD subunit of the premRNA cleavage
and polyadenylation specificity factor. The Dictyostelium PdeE gene is
expressed at highest levels during aggregation and its disruption causes
the loss of a cAMP-phosphodiesterase activity. The pdeE null mutants show
a normal cAMP-induced cGMP response and a 1.5-fold increase of cAMP-induced
cAMP relay. Overexpression of a PdeE-YFP1 fusion construct causes inhibition
of aggregation and loss of the cAMP relay response, but the cells can
aggregate in synergy with wild-type cells. The PdeE-YFP fusion protein was
partially purified by immuno-precipitation and biochemically characterized.
PdeE and its Dictyostelium ortholog PdeD are both maximally active at pH 7.0.
Both enzymes require bivalent cations for activity. The common cofactors Zn2+
and Mg2+ activated PdeE and PdeD maximally at 10 mM, while Mn2+ activated the
enzymes to 4-fold higher levels, with half-maximal activation between 10
and 100 M. PdeE is an allosteric enzyme, which is about 4-fold activated by
cAMP, with half-maximal activation occurring at about 10 M and an apparent
KM around 1 mM. cGMP is degraded at a 6-fold lower rate than cAMP. Neither
cGMP nor 8BrcAMP are efficient activators of PdeE activity.
submitted by Pauline Schaap [p.schaap@dundee.ac.uk]
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Differentiation-inducing factor-1 (DIF-1) inhibits STAT3 activity involved
in gastric cancer cell proliferation via MEK-ERK-dependent pathway.
Kanai M, Konda Y, Nakajima T, Izumi Y, Kanda N, Nanakin A, Kubohara Y,
Chiba T.
Kyoto University School of Medicine, Gunma University IMCR
Oncogene (2003) 22, 548-554.
Differentiation-inducing factor-1 (DIF-1) is a chlorinated hexaphenone
isolated from Dictyostelium. DIF-1 exhibits antitumor activity in several
types of mammalian tumor cells, although the underlying mechanisms remain
unknown. On the other hand, recent studies indicate that constitutively
activated STAT3 acts as an oncogene and could be a target for antitumor
drug. In the present study, we examined the effects of DIF-1 on
proliferation of gastric cancer cell lines as well as on its signal
transduction pathways, focusing mainly on STAT proteins. DIF-1 inhibited
proliferation of gastric cancer cells. Western blot analysis and
electrophoretic mobility shift assay showed that DIF-1 inhibited STAT3
activity in an MEK-ERK-dependent manner in gastric cancer cell lines,
AGS and MKN28. Moreover, blockade of STAT3 activity by ectopic expression
of dominant-negative STAT3 or the Janus kinase inhibitor, tyrphostin
AG490, inhibited cell growth of AGS cells. These results suggest that
STAT3 activity plays an important role for cell growth in AGS cells,
and raises the possibility that inhibition of STAT3 activity is one of
the mechanisms responsible for the antitumor effect of DIF-1 in these
cells.
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[End Dicty News, volume 20, number 2]
